Therapeutic use of anti-tf-antigen antibody

ABSTRACT

The invention is related to administration of monoclonal antibody JAA-F11 to an individual for inhibition of metastasis and/or inhibition of growth of cells which express TF—Ag, or for detection of tumors or metastatic foci which express TF—Ag. For inhibition of metastasis or inhibition of growth of cells expressing TF—Ag, the method comprises administering to the individual a therapeutic amount of mAb JAA-F11, wherein the JAA-F11 mAb inhibits the metastasis and/or growth of the TF—Ag expressing cancer cells. For detection of tumors or metastatic foci, mAb JAA-F11 is conjugated to a detectable label and administered to the individual. Detection of the label identifies metastatic foci or tumors which comprise cancer cells expressing TF—Ag.

This application is a Divisional of U.S. application Ser. No.11/190,165, filed on Jul. 26, 2005, which in turn claims priority toU.S. provisional application No. 60/591,011, filed on Jul. 26, 2004, thedisclosures of each of which are incorporated herein by reference.

This invention was supported by grant number R15AI49210-01 from theNational Institutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates generally to the inhibition of cancer cellgrowth and metastasis. More particularly, the invention relates to theuse of a monoclonal antibody to inhibit metastasis of cancer cells in anindividual.

BACKGROUND OF THE INVENTION

During carcinogenesis, alterations occur in the biosynthesis ofcarbohydrate structures on the cell surface, and several differentcarbohydrates linked either to proteins or to lipids have beenrecognized to be tumor-associated antigens. TF-Antigen (TF—Ag) is atumor-associated antigen of several carcinomas, including breast, lung,bladder, prostate and pancreas (1-4). TF—Ag has been proposed to beinvolved in the metastatic process (5-10).

TF—Ag is Galactosep 1-3 N-Acetyl Galactosamine, a disaccharide attachedto a protein by an alpha O-serine or O-threonine linkage. TF—Ag wasdiscovered by Thomsen, Friedenreich and Hueber in the late 1920's (2).TF—Ag is hidden on various normal cell membranes because it is linked toother carbohydrates, either by tertiary structures or by highlynegative-charged sialic acid (3,7). The densities of TF—Ag predict thehistologic grade of carcinomas (9,11), the invasive potential, and theprobability of early recurrence in breast (2, 12-13), urinary bladder,and prostatic carcinoma (14).

TF—Ag is more than an immunopathologic marker; it is postulated to havea role in adhesion and metastasis (2). Reversible or irreversibleadhesion is a primary step of invasion (15) and may occur when the TF—Agadhesion molecules recognize ligands such as galectins or other lectins(4,16). There is an increased expression of TF—Ag in metastatic tumors,and lectins that bind TF-AG are in common sites of metastatic tumorgrowth (17). Ligands for TF—Ag adhesion have been found in the vascularendothelium, the liver, the bone marrow and the lymph nodes (18-19) andthis may explain how TF—Ag levels are related to carcinomaaggressiveness (20).

There have been reports concerning the immunotherapeutic value of aninduced immune response to carbohydrate tumor associated antigens (8,26-29). For example, O'Boyle et al show development of very low levelIgG and IgM responses to two antigens which are related to TF-Ag, Tn(GalNAc) and sialylated Tn (NANAα2-6GalNAc) in colon cancer patients(26-27). Longenecker's studies in breast cancer patients used a keyholelimpet hemocyanin (KLH) conjugate of sialyl Tn and obtained higherresponses and observed some clinical response in these patients (29-30).Springer and Desai used vaccination with a T/Tn vaccine composed oftypes O and MN red blood cell derived glycoproteins which resulted inimproved breast cancer patient survival although only small amounts ofIgM were made (15). Immunization of breast cancer patients with Globo H(a hexasaccharide which contains TF—Ag), conjugated to KLH injected withthe adjuvant QS21 to improve immunogenicity resulted in most patientsforming only an IgM response (31). However, IgM is generally of loweraffinity and specificity than IgG, and represents a less mature immuneresponse, and many previous studies relating to antibodies to TF—Aginvolve IgM antibodies. Further, some anti-TF—Ag antibodies are notclinically useful because they cause undesirable proliferation of tumorcells (45). Moreover, while peptides which can bind to TF—Ag and inhibitcell adhesion in vitro have been described (5), it has also been foundthat some peptides which can bind to the TF—Ag on human carcinoma celllines and interfere with cell aggregation exhibit a low monomericaffinity for the TF—Ag, and therefore cannot be dissolved at highconcentration in aqueous buffers, which significantly limit theirpotential use (Landon et al., Journal of Protein Chemistry, (2003) Vol.22: pp 193-204).

One study purported to investigate the effect of a monoclonal antibodyto TF—Ag on cancer cells in a mouse model (Shigeoka, et al. TumorBiology (1999) 20: 138-146). However, that study attempted to simulatemetastasis by direct injection into the liver of cancer cells that werepre-incubated with the monoclonal antibody before being injected. Noduleformation in the livers of mice injected with the cells pre-incubatedwith the monoclonal antibody were compared to mice receiving injectionswith cancer cells that were not pre-incubated with the monoclonalantibody. While there were fewer nodules in mice receiving thepre-incubated cells, this study did not provide a relevant model ofantibody-mediated inhibition of metastasis because, due to thepre-incubation of the cancer cells with the monoclonal antibody, therewas no requirement for the antibody to travel through the body to locateand bind to cells expressing the TF—Ag. Further, there was no assessmentas to whether the nodule formation was the result of cancer cellstraveling through the endothelium in a manner similar to the naturalmetastatic process or whether the nodules were simply the result oflodging and proliferation of the injected cells.

Finally, while the production and characterization of another anti TF—Agmonoclonal antibody has been described (21), none of the aforementionedstudies demonstrate that the use of a monoclonal antibody to TF—Ag wouldinhibit either metastasis or growth of cancer cells in an individual.

SUMMARY OF THE INVENTION

In the present invention is provided a method for inhibiting metastasisof cancer cells in an individual, a method for inhibiting the growth ofcancer cells in an individual and a method for detecting metastatic focior tumors in an individual. The methods are related in that they entailthe use of a monoclonal antibody (mAb) JAA-F11 to target cancer cells invivo, which cancer cells express TF—Ag molecules.

In this regard, the method of inhibiting metastasis comprisesadministering to the individual a therapeutic amount of mAb JAA-F11,wherein the JAA-F11 mAbs inhibit the metastasis of TF—Ag expressingcancer cells. JAA-F11 mAbs may be administered alone, in combinationwith chemotherapeutic agents to which the mAbs are not conjugated, orconjugated to a chemotherapeutic agent. For example, JAA-F11 mAbs may beconjugated to any of a variety of enzymatically active toxins andfragments thereof, or to cytotoxic radioisotopes.

The method for inhibiting the growth of cancer cells in an individualcomprises administration of a therapeutic amount of mAb JAA-F11 to theindividual, wherein the JAA-F11 mAbs inhibit the growth of cancer cellswhich express TF—Ag. As in the method for inhibiting metastasis, whenJAA-F11 mAbs are administered to inhibit the growth of cancer cellswhich express TF—Ag, the JAA-F11 mAbs may be administered alone, incombination with chemotherapeutic agents to which the mAbs are notconjugated, or conjugated to chemotherapeutic agents.

In another embodiment is provided a method for identifying in anindividual metastatic foci, tumors, or combinations thereof. The methodcomprises the steps of administering to the individual JAA-F11 mAbs,wherein the mAbs have been conjugated to a detectable label, anddetecting the detectable label to identify the metastatic foci ortumors. For example, the mAbs may be conjugated to a spin label for usein magnetic resonance imaging (MRI).

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the regions of TF—Ag determinedto be important for binding to JAA-F11.

FIG. 2 is a graphical representation of results demonstrating theinhibitory effect of JAA-F11 on breast cancer cell adhesion toendothelium. Experimental results showed the inhibitory effect ofJAA-F11 on MDA-MB-435 metastatic human breast carcinoma cell lineadhesion to the endothelium. In FIGS. 2A and 2B, the inhibition of theadhesion to HUVEC monolayer (FIG. 2A) and human bone marrow endothelialcells HBMEC-60 (FIG. 2B) in static adhesion experiments is shown. InFIG. 3C, the effect of JAA-F11 on MDA-MB-435 rolling inwell-differentiated microvessels of perfused porcine dura mater ex vivois shown. In the graphs, * designates P<0.05 and ** P<0.01 respectively.

FIG. 3 is a graphical representation of results from a whole cellindirect enzyme immunoassay (EIA) and depicts reactivity of five mousecancer cells at five different JAA-F11 dilutions. This demonstrates that4T1 and JC cells are the highest expressors of TF—Ag, followed by LL andRIF, and the myeloma cells are either negative or very low for TF—Agexpression.

FIG. 4 is a graphical representation of results from cells grown withJAA-F11 antibody relative to cells grown without antibody in an MTTassay (41-42). Cell lines 4T1, JC, Lewis Lung, and RIF (all TF—Ag⁺),show significant inhibition of growth as indicated by the asterisk whilethe control cells (Myeloma) do not exhibit growth inhibition.

FIG. 5 is a graphical representation of results from a Kaplan-Meiersurvival curve generated from an in vivo experiment using mice (Group 1:PBS control; Group 2: JAA-F11 treatment). The results show that there isa significant (p=0.05) survival advantage to treatment with JAA-F11.

FIG. 6A is a photographic representation of lungs from PBS treated andJAA-F11 treated mice. FIG. 6A shows lungs from mice in the PBS controlgroup, and FIG. 6B shows lung from mice in the JAA-F11 group. Arrowspoint to the metastatic foci. The photographs demonstrate that there isa significant decrease in the number of visible metastatic foci on thelungs of JAA-F11 treated mice.

FIG. 7 is a graphical representation of inhibition of metastasis resultsfrom PBS treated and JAA-F11 treated mice.

DESCRIPTION OF THE INVENTION

In the present invention is provided a method of inhibiting metastasisof cancer cells in an individual, which cancer cells express TF—Agmolecules. The method comprises administering to the individual atherapeutic amount of JAA-F11 mAbs (ATCC Catalog number CRL-2381),wherein administration of the JAA-F11 mAbs inhibits the metastasis ofthe TF—Ag expressing cancer cells.

In another embodiment, a method is provided for inhibiting in anindividual the growth of cancer cells which express TF—Ag. The methodcomprises administering to the individual an amount of JAA-F11 mAbseffective to inhibit the growth of cancer cells expressing TF—Ag.

In another embodiment is provided a method for identifying in anindividual metastatic foci, tumors, or combinations thereof, wherein themetastatic foci or tumors comprise cells expressing TF—Ag. The methodcomprises the steps of administering to the individual JAA-F11 mAbs,wherein the JAA-F11 mAbs have been conjugated to a detectable label, anddetecting the detectable label to identify metastatic foci, tumors, orcombinations thereof.

We have determined that mAb JAA-F11 does not bind to GM1, a glycolipidexpressed on the surface of many cell types, including the centralnervous system. This is in contrast to a previously described mAb toTF—Ag, termed “170H82,” which binds to TF—Ag and GM1 (22). Therefore,this broader reactivity of the 170H82 mAb would result in reaction withnormal tissues that contain GM1, whereas mAb JAA-F11 does not. Thus, mAbJAA-F11 is more specific for cancer cells than other TF—Ag mAbs.Further, we have also determined that JAA-F11 does not causeproliferation of cells, unlike other anti-TF—Ag antibodies that havebeen reported (45).

In one embodiment, the present invention contemplates making a“humanized” mAb JAA-F11 for use in the method of inhibiting metastasisof cells which express TF—Ag or in the method of inhibiting the growthof cells which express TF—Ag. “Humanized” forms of non-human (e.g.,mice) antibodies are chimeric antibodies that contain minimal sequencederived from the non-human antibody. Humanized antibodies areessentially human immunoglobulins (also called the “recipient” antibody)in which residues from a hypervariable region of the recipient arereplaced by residues from a hypervariable region of a non-human species(also called a “donor” antibody) such as mouse, rat, rabbit or non-humanprimate having the desired antibody specificity, affinity, andcapability. In some instances, framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also can comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art.Humanization of mAb JAA-F11 can be essentially performed following themethod of Winter and co-workers by substituting mouse CDR sequences forthe corresponding sequences of a human antibody (Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)).

In another embodiment, an antigen-binding or variable region fragment ofmAb JAA-F11 may be used in the method of the invention. Examples ofsuitable antibody fragments include mAb JAA-F11 Fab, Fab′, F(ab′)₂, andFv fragments. Various techniques have been developed for the productionof antibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al,Journal of Biochemical and Biophysical Methods 24:107-117 (1992) andBrennan et al., Science, 229:81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. For example, theantibody fragments can be isolated from the antibody phage libraries.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)2 fragments (Carter et al.,Bio/Technology 10: 163-167 (1992)). According to another approach,F(ab′)2 fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of mAb JAA-F11 fragmentswill be apparent to the skilled practitioner.

In another embodiment, JAA-F11 mAbs may be conjugated to achemotherapeutic agent to enable localization of the chemotherapeuticagent to cancer cells via binding of the conjugated JAA-F11 mAbs tocells expressing TF—Ag. Chemotherapeutic agents useful in the generationof such antibody conjugates include enzymatically active toxins andfragments thereof. Suitable enzymatically active toxins includediphtheria A chain, nonbinding active fragments of diphtheria toxin,exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alpha sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes.

Conjugates of the JAA-F11 mAbs and chemotherapeutic agents may be madeusing a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyriyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared essentially as described in Vitetta et al. Science 238: 1098(1987).

In another embodiment, the JAA-F11 mAbs may be conjugated to aradioactive agent. A variety of radioactive isotopes are available forconjugating to JAA-F11 mAbs such that cells to which the JAA-F11 mAbsbind may be imaged or selectively destroyed. For selective destructionof cells expressing TF—Ag, the JAA-F11 mAbs may be conjugated to ahighly radioactive atom, such as In¹¹¹, At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶,Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.

When the JAA-F11 mAb conjugates are used for identifying cellsexpressing TF—Ag in metastatic foci or in tumors, the JAA-F11 mAbsconjugates may comprise a radioactive atom for scintigraphic studies,for example Tc^(99m) (metastable technetium-99), I¹²³, or a spin labelfor nuclear magnetic resonance (NMR) imaging (also known as magneticresonance imaging, or “MRI”), such as I¹²³, I¹³¹, I¹²⁴, F¹⁹, C¹³, N¹⁵,O¹⁷ or Gadlinium (III) or Manganese (II).

The radio-labels may be incorporated in the JAA-F11 mAbs in known ways.For example, labels such as tc^(99m) or I¹²³, Re⁸⁶, Re¹⁸⁸ and In¹¹¹ canbe attached via a cysteine residue in the JAA-F11 mAbs. Y⁹⁰ can beattached via a lysine residue. The Bolton Hunter method can be used toincorporate I¹²³. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal,CRC Press 1989) describes suitable methods in detail.

The use of antibodies for identification of metastatic foci and/ortumors by in vivo imaging is well known in the art. For example,antibody-chelators labeled with In¹¹¹ have been described for use in theradioimmunoscintographic imaging of carcinoembryonic antigen expressingtumors (Sumerdon et al. Nucl. Med. Biol. 1990 17:247-254). Inparticular, these antibody-chelators have been used in detecting tumorsin patients suspected of having recurrent colorectal cancer (Griffin etal. J. Clin. One. 1991 9:631-640). Antibodies with paramagnetic ions aslabels for use in MRI have also been described (Lauffer, R. B. MagneticResonance in Medicine 1991 22:339-342). Suitably labeled JAA-F11 mAbscan be used in a similar manner.

Labeled JAA-F11 mAbs can be injected into patients diagnosed with orsuspected of having a metastatic disease to identify metastatic fociand/or tumors. Information from such imaging can be used for diagnosingor staging of the disease status of the patient. The label used can beselected in accordance with the imaging system to be used. For example,Indium¹¹¹, Technetium⁹⁹ or Iodine¹³¹ can be used for planar scans orsingle photon emission computed tomography (SPECT). Positron emittinglabels such as Fluorine¹⁹ Iodine¹²³ and Iodine¹²⁴ can be used inpositron emission tomography. Paramagnetic ions such as Gadlinium (III)or Manganese (II) can used in magnetic resonance imaging (MRI).Localization of the label within a particular tissue of the individualpermits localization of metastatic foci or tumors which comprise cellsexpressing TF—Ag. A concentration of label at a particular locationgreater than background permits identification of the presence ofmetastasized cells. In a preferred embodiment, after administration oflabeled JAA-F11 mAbs, a suitable period of time is allowed to pass suchthat unbound JAA-F11 mAbs are cleared from the individual such thatbackground label is greatly reduced.

Therapeutic formulations comprising conjugated or unconjugated JAA-F11mAbs may be prepared by mixing with pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The JAA-F11 mAbs may be administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, intralymphatic or subcutaneousadministration. In addition, the JAA-F11 mAbs may suitably beadministered by pulse infusion, e.g., with declining doses of theantibody. Preferably, the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic. In preferred embodiments, theJAA-F11 mAbs are administered to an individual diagnosed with orsuspected of having breast, colon, prostate, ovarian, bladder or otherTF Ag⁺ cancers to inhibit metastasis or to inhibit the growth of thecancer cells.

One may also administer other compounds, such as chemotherapeuticagents, immunosuppressive agents and/or cytokines with the JAA-F11 mAbs.The combined administration can include co-administration, usingseparate formulations or a single pharmaceutical formulation, and canalso include consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities.

The JAA-F11 mAbs may be administered to a human or other animal inaccordance with the aforementioned methods of treatment in an amountsufficient to inhibit metastasis and/or growth of cells expressingTF—Ag. The JAA-F11 mAbs can be administered to such human or otheranimal in a conventional dosage form prepared by combining the JAA-F11mAbs with a conventional pharmaceutically acceptable carrier or diluentaccording to known techniques. It will be recognized by one of skill inthe art that the form and character of the pharmaceutically acceptablecarrier or diluent is dictated by the amount of active ingredient withwhich it is to be combined, the route of administration and otherwell-known variables, such as the size of the individual and the stageof the disease.

Dosages of radiolabeled JAA-F11 mAbs will also vary depending on thepatient, the antibody specificity, half-life, radioisotope stability,etc., and the extent of disease. Such dosages can be determined by oneskilled in the art. In one embodiment, a dosage of 4 mg/kg body weight,or a maintenance dose of 2 mg/kg body weight can be used.

The following Examples are meant to illustrate the invention and are notmeant to limit the scope of the claims.

EXAMPLE 1

This Example demonstrates the inhibition of cancer cell adhesion toendothelial and bone marrow cells by monoclonal antibody JAA-F11. Humanumbilical vein endothelial cells (HUVEC) were purchased from CascadeBiologics (Portland, Oreg.). The basal Medium 200 (Cascade Biologics)supplemented with low serum growth supplement (LSGS) containing FBS (2%v/v final concentration), hydrocortisone, human fibroblast growthfactor, heparin, and human epidermal growth factor was used for theHUVEC. The cells at passages 8-12 were used for the adhesionexperiments. The human bone marrow endothelial cell line, HBMEC-60,provided by Dr. C. E. van der Schoot (University of Amsterdam,Amsterdam, the Netherlands), was developed by immortalization of HBMECoriginally isolated from adult human bone marrow using the amphotrophichelper-free retrovirus pLXSN16 E6/E7 (32). The HBMEC-60 cells were shownto maintain their normal phenotype and adhesive properties, specificallythe ability to bind haematopoietic progenitor cells (32). The basalMedium 200 (Cascade Biologics) supplemented with 20% FBS and LSGScontaining hydrocortisone, human fibroblast growth factor, heparin, andhuman epidermal growth factor was used for HBMEC-60. Cells weremaintained in monolayer culture in a humidified incubator (5% CO₂) at37° C.

Adhesion experiments using these cells were performed essentially aspreviously described (4-5). Briefly, a single cell suspension of cancercells pre-labeled with 3 μg/ml of acridine orange (5*10⁴ cells perchamber in 2.5 ml of complete media supplemented with the antibodytested) was added to the monolayer of the endothelial cells grown toconfluence directly on microscope slides using 4-well chamber slides(NalgeNunc, Naperville, Ill.). The chambers were sealed, and cells wereallowed to adhere for 1 h at 37° C., after which the chambers wereinverted for 30 min to allow sedimentation of nonadherent cells. Next,the medium was drained, samples were gently rinsed with PBS, fixed for30 min in 2% formaldehyde solution in PBS, mounted under cover glass,and examined by fluorescent microscopy. Four random fields in each wellwere photographed at 250× magnification and the total number of adheredcells in every field was counted. The assay was performed inquadruplicate for each condition. The results depicted in FIG. 2A andFIG. 2B demonstrate the inhibition of adhesion to HUVEC monolayer andhuman bone marrow endothelial cells HBMEC-60, respectively.

EXAMPLE 2

This Example demonstrates that monoclonal antibody can block a key stageof metastasis and metastatic tumor formation in an ex vivo mode. Toperform these experiments, perfused porcine dura mater was used inadhesion experiments as previously described (33-34). Briefly, duramater corresponding to one hemisphere, collected from mature femaleYucatan miniature swine (Charles River, Me.) within 30 min after animalssacrifice in accordance with the University of Missouri approved animalcare protocol, was dissected and flattened onto a Sylgard-coated 100 mmdish. A major branch of the median meningeal artery was cannulated, anddura vasculature was perfused at 15 μl/min first with Krebsphysiological salt supplemented with 1.0 mg/ml porcine serum albumin for20 min, then with vessel-labeling solution (0.3 μg/ml acridine orange inRPMI-1640 supplemented with 10% FBS and 1.0 mg/ml porcine albumin) foran additional 40 min. Prior to injection, cancer cells were pre-labeledfor 5 min with 3 μg/ml acridine orange solution in RPMI-1640 medium,rinsed three times, dissociated from plastic, pipetted to produce asingle cell suspension, filtered through a 20 μm nylon mesh to removecell clumps, and adjusted to 5·10⁴ cell/ml. Interactions of cancer cellswith dura microvasculature were monitored and video recorded at 30frames per second using a fluorescence video microscopy system based ona Laborlux 8 microscope (Leitz Wetzlar, Germany) equipped with 75-wattxenon lamp and a high sensitivity CCD video camera (COHU, San Diego,Calif.). For subsequent frame-by-frame analysis, the recorded analogvideo images were digitized using a media converter DVMC-DA2 (Sony,Japan) and Adobe Premier 6 software (Adobe Systems, San Jose, Calif.).

The results from these experiments are depicted in FIG. 2C anddemonstrate that JAA-F11 blocks human breast tumor cell rolling onporcine dura mater, which is a key stage in the formation of metastatictumors.

EXAMPLE 3

This Example demonstrates TF—Ag detection on tumor cell lines withJAA-F11 using Indirect Cellular ELISA. To perform these experiments,five mouse cancer cell lines were tested. 4T1 (ATCC Number: CRL-2539)and JC (ATCC Number: CRL-2116) are both from BALB/c strain mammary glandadenocarcinomas. The 4T1 cell line is an animal model for stage IV humanbreast cancer (35-37). When injected into BALB/c mice, 4T1 spontaneouslyproduces highly metastatic tumors that can metastasize to the lung,liver, lymph nodes and brain while the primary tumor is growing in situ(35-37). JC also are able to produce tumors in BALB/c mice (38). Myeloma(ATCC Number: CRL-1580) is the fusion partner for producing JAA-F11hybridoma (21), and was used as a TF—Ag-negative control cell line. LL(ATCC Number: CRL-1642) is the Lewis lung carcinoma cell line also frommouse (39-40). The RIF cell line is a radiation-induced fibrosarcoma.Except for the culture medium for LL (Dulbecco's modified Eagle's medium(DMEM) with 4 mM L-glutamine with 1.5 g/L sodium bicarbonate and 4.5 g/Lglucose (90%) plus 10% FBS), the other four cell lines were grown inRPMI 1640 medium with 2 mM L-glutamine with 1.5 g/L sodium bicarbonate,4.5 g/L glucose, 10 mM HEPES and 1.0 mM sodium pyruvate (90%) plus 10%FBS.

To harvest and prepare single-cell suspensions, 4T1, JC, LL and RIF weretrypsinized for removal from the flasks. The cell suspensions wereplaced into 5 ml tubes in triplicate. Four percent formaldehyde wasadded and incubated for 20 minutes to fix cells. The tubes werecentrifuged and the supernatants were decanted. PBS-Tween-1% BSA wasadded back to each tube to prevent the fixed cells from drying. Thetubes with fixed cells were stored at 4° C. overnight or up to twoweeks. A peroxidase-linked immunoassay was performed in tubes. DifferentJAA-F11 antibody dilutions were added to each tube and incubated for 2hours at 37° C. After the incubation, the tubes were washed three timeswith 3 ml wash buffer (PBS-Tween, no azide). Anti-mouse IgG (γ-chainspecific) peroxidase conjugate at 1:2000 in PBS-Tween-1% BSA was addedto each tube, incubated 1 hour at RT, and washed three times.O-phenylenediamine dihydrochloride (Sigma, St. Louis Mo.) was added andafter one hour, 100 μl of the stop solution (1 N H₂SO₄) was added toeach tube followed by centrifugation for 10 min at 1200 rpm. 200 μl ofwas transferred into the respective wells in a microtiter plate and theplate was read on a microplate reader at 490 nm, using a well ofunreacted substrate as a blank to zero the reader.

The JC and 4T1 (breast cancer) and LL (lung cancer) cell lines wereexpected to be positive, and the RIF (fibrosarcoma) cell line and the M(myeloma) cell line were used as negative controls. The binding ofvarious concentrations of JAA-F11 mAb to these cells was measured. Theresults depicted in FIG. 3 demonstrate reactivity of the five mousecancer cells at five different JAA-F11 dilutions. The reaction withJAA-F11 was linear. This reaction was also linear with cell number (datanot shown). Myeloma cells, as expected, were shown to be negative forTF—Ag expression.

These results demonstrate that mAb JAA-F11 can be used to selectivelybind to cancer cells that express TF—Ag, and that breast cancer cells(4T1 and JC) and lung cancer cells (LL) cells are very high expressersof TF—Ag.

EXAMPLE 4

This Example demonstrates the effect of monoclonal antibody JAA-F11 oncell growth. To determine the direct effect of JAA-F11 on tumor cellgrowth, an in vitro proliferation analysis was performed (41-42). 4T1,Lewis Lung, JC, RIF and Myeloma cells were grown as described in Example3. The cells were plated at a concentration which was still in thelinear portion of the growth curve at 72 hours. Fifty μg/ml of JAA-F11mAb was added to each of 8 wells of cells for co-culture. Cells inculture medium without Ab and culture medium alone were used as negativecontrols. After 68 hours of cell growth in 96 well plates at 37° C., 5%CO₂, 10 μl of the tetrazolium salt3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide: thiazoylblue (MTT) was added to each well of the culture plate and the plateswere returned to the incubator for 4 hours, for a total growth time of72 hours (41-42). At the end of the 72 hr incubation period, theresulting insoluble formazan product in each well was solubilized byadding 120 μl of 5% formic acid in isopropanol with forceful mixing. Theabsorbance of each well was measured with a microplate reader at 570 nmas an indication of the metabolic activity of the cells.

After performing the assay on three separate days in sets of 8 on eachday, an average optical density (O.D.) was obtained. The average of thecells grown with JAA-F11 was statistically compared to cells grownwithout JAA-F11. A statistically significant decrease in cell growth dueto the presence of JAA-F11 was seen in the cell lines found to be TF—Agpositive in Example 3; i.e., 4T1, JC, LL, and RIF (p<0.05), but not inthe TF—Ag negative myeloma cells. These results are summarized in FIG.4. The results are expressed as a comparison of the amount of cellsgrowing with JAA-F11 mAb to the same number of cells grown withoutJAA-F11 mAb.

Thus, this Example demonstrates that JAA-F11 has a statisticallysignificant inhibitory effect (approximately 20%, p<0.01, which variedby cell type) on in vitro tumor cell growth, unlike other Abs to TF—Ag,which enhance in vitro tumor growth.

EXAMPLE 5

This Example demonstrates an in vivo anti-metastatic effect ofmonoclonal antibody JAA-F11. To perform these experiments, the 4T1 mousebreast cancer model was used. The 4T1 tumor line has severalcharacteristics that make it a suitable experimental animal model forhuman mammary cancer. First, 4T1 tumor cells are TF—Ag positive, easilytransplanted into the mammary gland so that the primary tumor can growin situ, and metastases readily develop. The primary tumor can besurgically removed, so that metastatic disease can be studied in themodel, comparable to the clinical situation where the primary breasttumor is surgically removed and metastatic foci may remain intact(35-37).

4T1 tumor cells were cultured with RPMI 1640 medium containing 2 mML-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPESand 1.0 mM sodium pyruvate (90%) plus 10% FBS in a 37° C., 5% CO₂ tissueculture incubator. Cultures were split using standard trypsinizationprotocols 2 to 3 times per week and were not grown in vitro longer thanone month before implantation. According to previous studies, 1×10⁴viable tumor cells per mouse was selected to ensure the tumor incidencewill be 100% but the tumor load will be not too great for Ab treatment(35-37).

One hundred microliters of 105 viable cells/ml 4T1 cell suspension wassubcutaneously inoculated in the right abdominal mammary gland ofeight-week-old female BALB/c mice. Three days after implanting the 4T1tumor cells, twenty mice were divided into two groups randomly, andreceived an intraperitoneal administration of either purified JAA-F11mAb (120 μg/100 μl/mouse) as treatment or PBS as control twice weekly.

On the fourteenth day after implanting 4T1 tumor, the primary breasttumors were surgically removed. The mouse weights were measured and theweight loss rates were compared between the treated and control mice.Clinical symptoms, such as lack of grooming, rough coat, rapid andlabored breathing, and loss of mobility were monitored and recorded andused as indicators of morbidity. Mice were sacrificed when weight lossreached 20%, or when significant morbidity occurred. Daily observationswere made by both investigators and animal caretakers. The survival timewas analyzed using a Kaplan-Meier Survival Curve (MedCalc). The organsof interest (primary tumor, lung, liver, spleen, lymph node, and brain)were collected and immunohistochemical staining was performed. Briefly,the selected tissues were fixed in Z-fixative (Zinc Formalin:Formaldehyde 3.7%, Zinc Sulfate, obtained from Histology Core Lab) witha volume at least 20 times that of the specimen. After 24 hours, fixedtissues were sent to the Histology Service Laboratory to beparaffin-embedded, cut into sections, and placed onto glass slides. APeroxidase-linked M.O.M. Immunodetection Kit (Vector Labs, BurlingameCalif.) was performed to detect the expression of TF—Ag on the primarytumors and the metastasic lesions from our animal experiments. Thetissue sections were deparaffinized and rehydrated using standardprotocols. The endogenous peroxidase activity was blocked by incubatingthe tissue sections with 3% hydrogen peroxide in tap water for 5 min.The sections were washed twice for 2 minutes in PBS. The M.O.M. kit wasused as directed with purified JAA-F11 antibody (0.24 mg/ml) in M.O.M.Diluent for 1 hour used in the primary antibody step.

For in vivo immunotherapy after primary tumor removal, forty mice wereused, 20 receiving PBS as a control and 20 receiving JAA-F11. On thefourteenth day after implantation the primary breast tumors weresurgically removed. The weight changes of mice were measured twice aweek and the mice were sacrificed when the weight loss reached 20% orsignificant morbidity occurred.

The survival time was recorded and is depicted in FIG. 5. These datawere analyzed by the Kaplan-Meier Survival Curve (MedCalc). The mediansurvival time of the PBS and JAA-F11 groups were 57 and 72 daysrespectively. The difference was significant (P=0.05). The animals ofJAA-F11 treatment group lived significantly longer than the PBS controlgroup. The organs of interest, such as primary tumor, lung, liver,brain, and spleen were collected. Metastatic lesions were found on lymphnodes, ribs, pericardium, and lungs in mice from both groups.

Further, the JAA-F11 treated mice had significantly lower numbers ofgrossly apparent metastatic lesions in their lungs, with representativelungs shown in FIG. 6. The levels of metastases on lungs were classifiedinto four groups after counting the number of metastatic lesion grosslyvisible in the lungs (Table 1). The Chi-Square Test (MedCalc) wasperformed to detect if the frequencies of metastasis levels on lungsfrom PBS and JAA-F11 group were significantly different. As can be seenin Table 1, the difference between the JAA-F11 group and PBS group issignificant (P=0.0155). The mice receiving JAA-F11 treatment had lessmetastasis to the lungs than those receiving PBS control. The lungs thatwere grossly negative for tumor metastasis were examined formicrometastasis by a pathologist in a blinded study with several lungswith metastatic lesions. Of the two mice in the PBS control group “nometastases observed” group, one had histologically apparent tumornodules, while the other did not. Of the 10/19 sets of lungs retrievedfrom the JAA-F11 “no metastases observed” group, 2 were from mice thathad died due to tumor load, and these

TABLE 1 Summary of the Levels of Metastases on Lungs Mice in PBS Mice inJAA- # Metastases on lungs group F11 group +++ (>20) 6/16 3/19 (37.50%)(15.79%) ++ (10-20) 4/16 2/19 (25.00%) (10.53%) + (1-9) 4/16 4/19(25.00%) (21.05%) − (no metastases observed) 2/16 10/19  (12.50%)(52.63%)did have microscopic evidence of tumor. The lungs from the 8 mice thathad no evidence of disease at the time of the termination of theexperiment had no histological evidence of disease in their lungs, thusthey were negative for tumor development as analyzed by histologicalsectioning. Thus, JAA-F11 significantly prolongs survival of micereceiving the treatment (FIG. 5) and inhibits metastasis to lungs(P=0.016) (FIGS. 6 and 7, and Table 1).

In summary, the in vitro and in vivo experiments described hereindemonstrate that administration of mAb JAA-F11 can inhibit cell adhesionin models of human cancer cell metastasis, mAb JAA-F11 can significantlyinhibit metastasis to lungs, and prolong survival of mice afterimplantation of 4T1 breast cancer cells and resection of the primarytumor. This indicates administration of JAA-F11 mAb is therapeuticallyuseful in two ways: 1) through the traditional antibody mediatedselective killing of tumor cells and 2) by decreasing the ability of thetumor cells to metastasize by blocking TF—Ag to lectin adhesion inendothelium and elsewhere.

This invention has been described through examples presented above.Routine modifications to the methods and compositions presented hereinwill be apparent to those skilled in the art and are intended to bewithin the scope of the claims appended hereto.

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1. A method for identifying metastatic foci or one or more tumors in anindividual, wherein the metastatic foci or the one or more tumorscomprise cells expressing TF—Ag, comprising the steps of: a)administering to the individual monoclonal antibody JAA-F11, wherein themonoclonal antibody JAA-F11 has been conjugated to a detectable label;and b) detecting the detectable label to identify the metastatic foci orone or more tumors.
 2. The method of claim 1, wherein the detectablelabel is selected from the group consisting of iodine¹²³, iodine¹³¹,iodine¹²⁴, indium¹¹¹ and fluorine¹⁹.
 3. The method of claim 1, whereindetecting the detectable label is performed by magnetic resonanceimaging or single photon emission computed tomography.
 4. The method ofclaim 1, wherein the one or more tumor or the metastatic foci comprisecancer cells selected from the group consisting of breast cancer cells,lung cancer cells, prostate cancer cells and pancreas cancer cells.